Controlling small molecules to self-assemble into supramolecular complexes is pervasive among living things, which build complex structures for high-order functions necessary for life (Whitesides et al., (1991) Science. 254: 1312-1319; Capito et al., (2008) Science 319: 1812-1816). In the laboratory, this principle of self-assembly is also widely used to synthesize supramolecules and nano/microstructures (O'Leary et al., (2011) Nat. Chem. 3: 821-828; Gazit, E. (2010) Nat. Chem. 2: 1010-1011; Yang et al., (2008) Acc. Chem. Res. 41: 315-326). Recently, extensive efforts have been made to design and control small molecules with the propensity to self-assemble in living cells (Liang et al., (2010) Nat. Chem. 2: 54-60; Gao et al., (2012) Nat. Commun. 3: 1033; Adler-Abramovich et al., (2012) Nat. Chem. Biol. 8: 701-706; Williams et al., (2011) Biomaterials 32: 5304-5310; Ye et al., (2011) Angew. Chem. Int. Ed. 50: 2275-2279). In these examples, membrane-permeable small molecules are shown to enter cells and undergo self-assembly after activation by intended cellular targets, such as an enzyme. This recent progress has already generated promising applications such as imaging a proteolytic enzyme activity in subcellular locations, and has provided possible means to investigate and control self-assembly in the context of living cells. Further advances in achieving controlled self-assembly of small molecules in whole mammalian organisms would offer myriads of applications in biology and medicine such as controlled drug delivery (Vemula et al., (2011) J. Biomed. Mater. Res. A 97: 103-110), synthesis of new functional molecules, new ways for regulating cellular processes and molecular imaging in living subjects. Unfortunately, there are no previous examples of controlled self-assembly of synthetic small molecules at the level of whole mammalian organisms. The in vivo physiological environment—a much more complex and dynamic setting than cultured cells—demands high bioorthogonality and biocompatibility of the chemistry that controls self-assembly, and also presents significant challenges to characterize such a system.
Currently, a number of biocompatible reactions including Staudinger ligation (Saxon & Bertozzi (2000) Science 287: 2007-2010; Lin et al., (2005) J. Am. Chem. Soc. 127: 2686-2695), strain-promoted azide-alkyne cycloaddition 14,15, trans-cyclooctene/tetrazine cycloaddition (Devaraj et al., (2009) Angew. Chem. Int. Ed. 48: 7013-7016; Blackman et al., (2008) J. Am. Chem. Soc. 130: 13518-13519; Lang et al., (2012) Nat. Chem. 4: 298-304), and Pictet-Spengler ligation (Agarwal et al., (2013) Proc. Natl. Acad. Sci. U.S.A. 110: 46-51), have been developed for probing biological interactions in living systems, some of which have been shown to work in cells (Yusop et al., (2011) Nat. Chem. 3: 239-243; Chan et al., (2012) Nat. Chem. 4: 973-984), but very few can work in living organisms (Prescher et al., (2004) Nature 430: 873-877; Laughlin et al., (2008) Science 320: 664-667; Devaraj et al., (2012) Proc. Natl. Acad. Sci. U.S.A. 109: 4762-4767; Sletten & Bertozzi (2011) Acc. Chem. Res. 44: 666-67622-25). A new biocompatible reaction between free cysteine and cyanobenzothiazole (CBT) has been reported that can proceed in physiological conditions with a fast second-order rate constant (9.1 M−1s−1) (Ren et al., (2009) Angew. Chem. Int. Ed. 48: 9658-9662; Van de Bittner et al., (2013) J. Am. Chem. Soc. 135: 1783-1795). This reaction has been successfully applied to protein labelling (Devaraj et al., (2012) Proc. Natl. Acad. Sci. U.S.A. 109: 4762-4767) and protease activity imaging in living cells (Liang et al., (2010) Nat. Chem. 2: 54-60).
Recently, a few radiolabeled sulfonamide small-molecule caspase-3 inhibitors have been reported for PET imaging of caspase-3 in apoptotic cells (Nguyen et al., (2009) Proc. Natl. Acad. Sci. USA 106: 16375-16380; Zhou et al., (2009) Org. Biomol. Chem. 7: 1337-1348; Faust et al., (2007) J. Nucl. Med. Mol. Imaging. 51: 67-73; Reshef et al., (2010) J. Nucl. Med. 51: 837-840). However, a PET tracer that is mechanistically similar to the activatable fluorescent probes and can image caspase-3 activity with signal amplification has not been reported.
MRI probes that can specifically report on enzyme activity have become particularly attractive due to variations in enzyme expression levels in many diseases. The inherently low detection sensitivity of MRI has limited the development of enzyme activatable MRI probes for in vivo application. However, novel activatable MRI probes enable can high spatial resolution imaging of specific enzyme activity in vivo and are thus in high demand.
Recently, an activatable thulium-based paramagnetic chemical exchange saturation transfer (PARACEST) MRI probe and a paramagnetic relaxation-based 19F MRI probe have been reported to detect caspase-3 activity with high sensitivity in solution. However, a probe responsive to caspase-3 that employs gadolinium, one of the major clinically used sources of MRI contrast, has not yet been reported.